Sumary of Work: Non-typeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and respiratory tract diseases in adults. We previously demonstrated that detoxified lipooligosaccharide (dLOS)-protein conjugates from NTHi elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (Infect. Immun. 64:4047). In this past year, we established a chinchilla otitis media model and evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-TT (tetanus toxoid), dLOS-HMP (high molecular weight proteins from NTHi) or saline (control) in Freund's adjuvant, and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS of which 49% (19/39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all controls (19/19) and 56% (22/39) of the vaccinated animals during a period of 21 days (p < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (p < 0.01). The incidence of infection in the unchallenged ear or inner ear was 26% or 28% in the vaccine groups and 53% or 58% in the controls (p < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicated that active immunization with LOS based conjugates results in a significant in vivo reduction of the incidence of NTHi-induced otitis media in an art-recognized animal model of human otitis media.